Compositions and methods for prion decontamination

ABSTRACT

The invention relates to compositions and methods for prion degradation, decontamination or disinfection. The composition comprises an oxidizing agent, one or more proteases and a surfactant such as an ionic surfactant/detergent. The method comprises contacting a prion contaminated entity with a prion-degrading composition comprising an effective amount of an oxidizing agent, an effective amount of at least one protease, and an effective amount of a surfactant. The components of the composition may be contacted with a prion-contaminated entity sequentially or simultaneously using an aqueous composition. Typically at least two different proteases are used for optimal efficacy. Preferably the oxidizing agent comprises peracetyl ions or a source thereof. The invention also relates to kits comprising the various reagents.

This application claims the benefit of U.S. Provisional PatentApplication No. 60/854831, filed Oct. 27, 2006, and U.S. ProvisionalPatent Application No. 60/925177, filed Apr. 19, 2007.

FIELD OF THE INVENTION

The present invention relates to methods and reagents for use in priondecontamination. In particular, the invention relates to priondecontamination of surgical instruments, advantageously avoidingautoclaving.

BACKGROUND OF THE INVENTION

The persistence and resistance of the prion agents responsible for CJD(Creutzfeldt-Jakob disease) has raised fears about the possibility ofiatrogenic transmission following surgery. The prion diseases, whichinclude scrapie, atypical scrapie in sheep, BSE (Bovine SpongiformEncephalopathy) in cattle, CWD (Chronic Wasting Disease) in deer and CJDin humans are a novel group of transmissible, fatal neurodegenerativeconditions. The transmissible agent termed a prion is comprised largelyor solely of a conformational isomer of a normal cellular PrP^(C) prionprotein. The disease related conformer, designated PrP^(Sc), has severalunusual properties including resistance to proteolysis, detergentinsolubility and high thermal stability. These physical propertiescoupled to observations that PrP^(Sc) adheres strongly to surgical steeland other materials present problems in the cleaning and sterilisationof surgical instruments as prion infectivity is known to be resistant toconventional autoclaving.

In the absence of a pre-clinical diagnostic test for CJD, pre-surgicaltesting of patients is not possible. Although in a minority of caseswhere CJD is suspected or confirmed, used instruments can be quarantinedor destroyed. However, for the majority of procedures, new methods ofdecontamination are required. There are many ongoing efforts, includingthose by the UK Department of Health (e.g. Medical Research Council(MRC) Prion Unit, London, UK), attempting to address the problem ofiatrogenic CJD transmissions.

Standard autoclaving, and in some cases high temperature autoclaving to134° C., is the hospital standard for prion decontamination. However,conventional studies have shown survival of prions under autoclaveconditions (Taylor, D M., J. Hosp. Infect. 43:S69-S76 (1999); Jackson etal., J. Gen. Virol 86:869-878 (2005)). Clearly prions will graduallyaccumulate under these conditions. Even the most effective autoclavewill only sterilize to the degree that heat and steam penetrate thearticles being treated. This is not straightforward when dealing withsurgical sets comprising numerous complex instruments.

Taylor (Taylor D. M., supra) discloses the use of sodium hypochloritesolutions and 2M sodium hydroxide in prion inactivation. However, thereare problems with this approach such as incomplete inactivation andincompatibility with many medical devices. Furthermore, resistance ofprions to autoclaving is reported.

The WHO (World Health Organization) guidelines on prion decontaminationrecommend autoclaving and immersing contaminated instruments in 1M NaOHand/or 20,000 ppm NaOCl (WHO report “Infection Control Guidelines forTransmissible Spongiform Encephalopathies”, Mar. 23-26 1999, Geneva,Switzerland, WHO/CDS/CSR/APH/2000.3). This is an extremely hazardousprocedure and can leave undesirable salt residues on surfaces.Furthermore, in addition to the safety aspects, the corrosive effect ofsuch alkali or oxidizing halogen species at that concentration, combinedwith the temperatures and pressures implicit to autoclaving, would belikely to destroy or at least seriously damage delicate surgicalinstruments.

Commercial reagents currently in use for cleaning of surgicalinstruments prior to autoclaving have little or no effect upon PrP^(Sc)contamination. Existing methods of decontamination such as thoseinvolving LpH® and LpH®se (Steris, Inc. Mentor, Ohio), and Endozyme Plus(Ruhof Corp., Mineola, N.Y.) are of limited use in destroyinginfectivity. Furthermore, some reagents are incompatible with medicalmaterials such as Dracom polymer (polysulfone).

Fichet et al. (Lancet 364:521-526 (2004)) describe three methods fordisinfection of prion contaminated medical devices. Firstly theydescribe use of an enzymatic cleaner (KLENZYME®, Merck & Co., Inc.,Whitehouse Station, N.J.) with autoclaving at 121° C. Secondly theydescribe alkaline cleaner (HAMO™ 100 PID, Steris, Mentor, Ohio). Thethird method described is the only one said to be suitable for fragiledevices such as endoscopes and involves use of the alkaline cleaner onwet instruments followed by a dry vaporized hydrogen peroxide (VHP)treatment. Fichet et al. also disclose enzymatic cleaner followed by VHPtreatment as being very effective. There is no disclosure of thecomposition of the enzymatic cleaner. Peracetic acid is used and shownto be ineffective (100% onward transmission rate following treatment).

The present invention seeks to overcome problem(s) associated with theprior art by providing effective prion decontamination compositions andmethods.

SUMMARY OF THE INVENTION

An aqueous prion-degrading composition is provided comprising acombination of active reagents including an effective amount of anoxidizing agent, at least one protease, and an effective amount ofsurfactant that can be used for effective prion degradation (i.e. toreduce the titre of infective prions) of a prion-contaminated entity.Preferably, the prion-degrading composition further comprises aneffective amount of an ionic surfactant. Surprisingly, the oxidizingagent and protease enzyme combination provides a synergistic effect onprion decontamination.

An aqueous prion-degrading composition is also provided comprising (a)an effective amount of at least one oxidizing agent; (b) an effectiveamount of at least one first protease; (c) an effective amount of atleast one second protease wherein said second protease is different fromsaid at least one first protease; and (d) an effective amount of asurfactant.

In a preferred embodiment, the aqueous prion-degrading compositioncomprises two proteases selected from the group consisting of NEUTRASE®,ALCALASE®, PRONASE®, and Proteinase K. In a further preferredembodiment, the two proteases are NEUTRASE® and ALCALASE®.

In a preferred embodiment, the surfactant is sodium dodecyl sulfate(SDS).

In another embodiment, the aqueous prion-degrading composition furthercomprises an additional ingredient selected from the group consisting ofpH adjusters, buffering agents, chelating agents, corrosion inhibitors,peroxygen stabilizers, and mixtures thereof.

The present invention further provides methods by whichprion-contaminated entities such as medical instruments can bedecontaminated of prion infectivity. According to the methods of thisinvention, the prion-contaminated entity is contacted with thecomponents of the aqueous prion-degrading composition sequentially orsimultaneously. Simultaneous contact is highly preferred.

It has surprisingly been found that the methods of this invention resultin the potentiating of activity of the components of the prion-degradingcomposition, resulting in improvements in prion decontamination. Inparticular use of the oxidizing agent in combination with the proteasesdescribed below result in a synergistic effect.

In one embodiment, a method for prion decontamination or disinfection isprovided which comprises: (a) contacting a prion-contaminated entitywith at least one surfactant; (b) contacting the entity with anoxidizing agent, and (c) contacting the entity with at least oneprotease.

In a particular embodiment there is provided a sequential method forprion decontamination or disinfection of a prion-contaminated entitywhich comprises: (a) first contacting the entity to be decontaminatedwith a surfactant, and then (b) contacting the entity to bedecontaminated with an oxidizing agent, and then (c) contacting theentity with a protease.

In another embodiment, a method for prion decontamination ordisinfection of a prion-contaminated entity comprises: (a) firstcontacting the entity with a surfactant, and then (b) contacting theentity with an oxidizing agent, and then (c) contacting the entity witha first protease, and (d) contacting the entity with a second protease.

In another embodiment, a method for degrading prion particles isprovided comprising, (a) providing an aqueous prion-degradingcomposition comprising (i) an effective amount of at least one oxidizingagent; (ii) at least one first protease; (iii) at least one secondprotease wherein the second protease is different from the at least onefirst protease, and (iv) an effective amount of a surfactant; (b)contacting a prion-contaminated entity with the prion-degradingcomposition of (a) under suitable conditions whereby prion particles aredegraded.

In another embodiment, the proteases and the oxidizing agent arecontacted simultaneously with the entity to be decontaminated. In thisembodiment, the surfactant may be contacted with the entity with theproteases and the oxidizing agent or the surfactant may be contactedwith the entity in a separate step.

In a preferred embodiment, the two proteases are selected from the groupconsisting of NEUTRASE®, ALCALASE®, PRONASE®, and Proteinase K. In afurther preferred embodiment, the two proteases are NEUTRASE®, andALCALASE®.

The prion-contaminated entity can have a solid surface or be a fluid,such as a biological fluid. Solid surfaces include medical and dentalinstruments. In one aspect, the prion-contaminated entity is selectedfrom the group consisting of a biological waste, equipment used in foodprocessing equipment, an enclosure used to house animals, a medicalinstrument, a dental instrument, and countertops. Food processingincludes, for example, slaughterhouses and poultry-processingfacilities. In a preferred embodiment, the prion-contaminated entity isselected from the group consisting of prion-contaminated medicalinstruments. In another preferred aspect, the medical instrument is anendoscope or laparoscope. In yet another aspect, the medical instrumentis comprised of steel, plastic or a combination thereof. The medicalinstrument may also be comprised of other materials of constructiontypically found in medical devices and instruments.

In another aspect, the prion-degrading methods and compositions are usedto decrease and/or eliminate iatrogenic CJD.

In a preferred aspect, the methods for prion decontamination areperformed in a medical instrument sterilizer.

In another aspect, any of the methods of the present invention furthercomprise and preferably have as a final step, autoclaving theprion-contaminated entity.

In one preferred aspect, the present method is used for low temperaturedecontamination where autoclaving is avoided.

In another embodiment, a kit is provided which comprises: (a) a firstreagent comprising an oxidizing agent; (b) a second reagent comprisingat least one protease; and (c) a third reagent comprising a surfactant.

In a preferred embodiment, the second reagent in the kit comprises atleast two different proteases. In a preferred embodiment, the twoproteases are selected from the group consisting of NEUTRASE®,ALCALASE®, PRONASE®, and Proteinase K. In a further preferredembodiment, the two proteases are NEUTRASE® and ALCALASE®.

In another preferred embodiment, the first, second, and third reagentsare provided individually as solids. In a preferred embodiment, thefirst, second, and third reagents of the kit are provided as solidpowders.

In another embodiment, the kit further comprises an additional reagentselected from the group consisting of pH adjusters, buffering agents,chelating agents, corrosion inhibitors, peroxygen stabilizers andmixtures thereof, which may be supplied as additional kit reagent or maybe included as an reagent in one or more of the first, second, and thirdreagents.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 a and 1 b. Western blot analyses of the data generated inExample 2 showing the no detectable efficacy of PERASAFE™ Sterilant fordecontamination of prions (FIG. 1 a) as compared to combined treatmentwith PERASAFE™ Sterilant and proteases and SDS (FIG. 1 b), whereindecontamination was exhibited.

FIG. 2. A Western blot of the data generated in Example 4 showing theefficacy of decontamination using optimized enzyme concentrations forthe proteolytic degradation of PrP^(Sc) in conjunction with an oxidizingagent (PERASAFE™ Sterilant) at 40° C. Marker lane is labeled as M; thecontrol lane, C, contains 12 μL of a 10% w/v brain homogenate as acontrol. Six lanes, as identified by incubation times, were loaded with12 μL of 10% w/v brain homogenate treated at 40° C. for 2, 10, 20, 30,40, and 60 minutes.

FIG. 3. A graph of the Scrapie Cell Assay (the cell culture assaydescribed in Example 5). Various dilutions of brain homogenate frominfected animals with RML (Rocky Mountain Laboratories) strains ofprions are coated on wires. The wires are subjected to thedecontamination treatment. Treatments at 40° C. and 50° C. were assayed.Various dilutions (RML) were plotted against the number of tissueculture infectious units (TC IU).

FIG. 4. A Western blot of the data generated in Example 6 showing theefficacy of decontamination using optimized enzyme concentrations forthe proteolytic degradation of PrP^(Sc) using various concentrations ofSDS in the absence of the oxidizing agent (PERASAFE™ Sterilant) at 50°C. Marker lane is labeled as M. The control lane, C, contains 12 μL of a10% w/v brain homogenate as a control. Lanes were loaded with 12 μL of10% w/v brain homogenate treated at 0, 0.5, 1.0, 1.5, and 2.0% (w/v)SDS. The lanes are identified by the SDS concentration.

FIG. 5. A Western blot of the data generated in Comparative Example Ashowing the efficacy of decontamination using various ALCALASE®concentrations for the proteolytic degradation of PrP^(Sc) in theabsence of an oxidizing agent (PERASAFE™ Sterilant) at 50° C. Markerlane is labeled as M. The control lane, C, contains 17 μL of a 10% w/vbrain homogenate as a control. Lanes were loaded with 17 μL of 10% w/vbrain homogenate treated with various concentrations of ALCALASE®, andidentified by ALCALASE® concentrations of 1.5, 1.25, 1.0, 0.75, 0.50,0.25 and 0 mg/mL.

FIG. 6. A Western Blot of the data generated in Comparative Example Bshowing the efficacy of decontamination using various NEUTRASE®concentrations for the proteolytic degradation of PrP^(Sc) in theabsence of an oxidizing agent (PERASAFE™ Sterilant) at 50° C. Markerlane is labeled as M. The control lane, C, contains 17 μL of a 10% w/vbrain homogenate as a control. Lanes were loaded with 17 μL of 10% w/vbrain homogenate treated with various concentrations of NEUTRASE® andidentified by NEUTRASE® concentrations of 1.5, 1.25, 1.0, 0.75, 0.50,0.25 and 0 mg/mL.

FIG. 7. A Western blot of the data generated in Example 7 showing theefficacy of decontamination using various ALCALASE®/NEUTRASE®concentrations (based on a fold dilution from previous examples; 2, 4,8, 16, 32, 64-fold dilutions) for the proteolytic degradation ofPrP^(Sc) in the presence of an oxidizing agent (PERASAFE™ Sterilant) at50° C. Marker lane is labeled as M. The control lane, C, contains 12 μLof a 10% w/v brain homogenate as a control. Lanes labeled 64, 32, 16, 8,4, and 2 were loaded with 12 μL of 10% w/v brain homogenate and therespective dilutions of ALCALASE®/NEUTRASE®. For example, the lanemarked as 2 is a 2-fold dilution of the standard ALCALASE®/NEUTRASE®concentrations (i.e. 1.58 mg/mL ALCALASE® and 6.32 mg/mL NEUTRASE®).

FIG. 8. A Western blot of the data generated in Example 8 showing theefficacy of decontamination using various ALCALASE®/NEUTRASE®concentrations (based on fold dilutions of 4, 6.7, 8.3, 10, and 12.5).Each lane is identified by the dilution factor, shown as lane labels,for the proteolytic degradation of PrP^(Sc) in the presence of anoxidizing agent (PERASAFE™ Sterilant) at 50° C. Marker lane is labeledas M. The control lane, C, contains 12 μL of a 10% w/v brain homogenateas a control. Lanes labeled 4, 6.7, 8.3, 10 and 12.5 were loaded with 12μL of 10% w/v brain homogenate and the respective dilutions ofALCALASE®/NEUTRASE®.

FIG. 9. A Western blot of the data generated in Comparative Example Cshowing the efficacy of decontamination using components of PERASAFE™Sterilant formulation components in combination with 1% w/v SDS, 750μg/mL ALCALASE® and 3 mg/mL NEUTRASE® at 50° C. Marker lane is labeledas M. The control lane, C, contains 16 μL of a 10% w/v brain homogenateas a control. Lanes labeled (a) through (f) were loaded with 16 μL of10% w/v brain homogenate and the respective components as identified inComparative Example C.

FIG. 10. A Western blot of the data generated in Example 9 showing theefficacy of decontamination using various ALCALASE®/NEUTRASE®concentrations (based on fold dilutions of 4, 6.7, 8.3, 10, and 12.5).Each lane is identified by the dilution factor, shown as lane labels,for the proteolytic degradation of PrP^(Sc) in the presence of anoxidizing agent (PERASAFE™ Sterilant) at 50° C. This is a repeat of thework original performed in Example 8 (FIG. 8). Marker lane is labeled asM. The control lane, C, contains 12 μL of a 10% w/v brain homogenate asa control. Lanes labeled 4, 6.7, 8.3, 10, and 12.5 were loaded with 12μL of 10% w/v brain homogenate and the respective dilutions ofALCALASE®/NEUTRASE®.

FIG. 11. A Western blot of the data generated in Example 10 showing theefficacy of decontamination using various components of PERASAFE™Sterilant or dilutions of complete PERASAFE™ Sterilant relative to thestandard 1× formulation in combination with 1% w/v SDS, 330 82 g/mLALCALASE® and 1.2 mg/mL NEUTRASE® at 50° C. Marker lane is labeled as M.The control lane, C, contains 13 μL of a 10% w/v brain homogenate as acontrol. Lanes labeled (a) through (e) were loaded with 13 μL of 10% w/vbrain homogenate and the respective components as indicated in Example10.

FIG. 12. Western blot of the data generated in Example 11 showing theefficacy of 1× PERASAFE™ Sterilant+1% w/v SDS+ALCALASE®+NEUTRASE® fordecontamination of prions at 40° C. vs. 50° C. The control lane, C,contains 12 μL of a 10% w/v brain homogenate as a control. Lanes 2, 5,10, and 20 at each temperature indicate the reaction times. These laneswere loaded with 12 μL of 10% w/v brain homogenate treated at thetemperatures and times indicated.

FIG. 13. Graphical comparison of PrP degradation kinetics at 40° C. and50° C. from the Western blot of FIG. 12 and data generated in Example11. The data are best described by a double exponential decay due to thetwo main species of PrP present; PrP^(C) and PrP^(Sc). The K_(1/2)values are very similar for both 40° C. and 50° C. (29 seconds and 22seconds, respectively).

FIG. 14. Western blot of the data generated in Example 12 comparing theefficacy of other oxidizing agents when used in combination with 1% w/vSDS and 300 μg/mL ALCALASE®+1.2 mg/mL NEUTRASE®. The control lane, C,contains 12 μL of a 10% w/v brain homogenate as a control. Laneslabelled “A”, “B”, “C”, “H”, “N”, “P”, and “V”) were loaded with 12 μLof 10% w/v brain homogenate treated as described. C=control, A=16.2mg/mL PERASAFE™ Sterilant (1×), B and C=13 mg/mL PERASAFE™ Sterilant(0.8×), H=a 1:70 dilution of hypochlorite (pH 8), N=3.34 mg/mL NaDCC(Neochlor), P=30 mg/mL PROXITANE® (pH 6.5), and V=20 mg/mL VIRKON® SDisinfectant (pH 6).

FIG. 15. A graphical representation of the levels of immunoreactivityshown in FIG. 14 based on data generated in Example 12. The lanes shownin FIG. 14 were quantified by densitometry and are displayed below as apercentage of the control value.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a prion-decontaminating composition, methods forprion decontamination and a kit for use in decontaminating andprion-contaminated entity.

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions apply unless specifically stated otherwise.

As used herein, the term “comprising” means the presence of the statedfeatures, integers, steps, or components as referred to in the claims,but that it does not preclude the presence or addition of one or moreother features, integers, steps, components or groups thereof.

As used herein, the term “about” modifying the quantity of an ingredientor reactant of the invention or employed refers to variation in thenumerical quantity that can occur, for example, through typicalmeasuring and liquid handling procedures used for making concentrates oruse solutions in the real world; through inadvertent error in theseprocedures; through differences in the manufacture, source, or purity ofthe ingredients employed to make the compositions or carry out themethods; and the like. The term “about” also encompasses amounts thatdiffer due to different equilibrium conditions for a compositionresulting from a particular initial mixture. Whether or not modified bythe term “about”, the claims include equivalents to the quantities. In afurther embodiment, “about” means the referenced value within 10%,preferably within 5%, and most preferably within 1%.

As used herein, the terms “prion” and “prion particles” refer to aproteinaceous disease-causing agent responsible for a number ofdegenerative brain diseases in animals and humans. Examples ofprion-associated diseases include, but are not limited toCreutzfeldt-Jakob Disease (all forms including CJD, iCJD (iatrogenic),vCJD (variant), sCJD (sporadic), and familial (fCJD)),Gerstmann-Sträussler-Scheinker Syndrome, Fatal Familial Insomnia, andKuru. Examples of prion-associated diseases in animals include, but arenot limited to scrapie, atypical scrapie in sheep, BSE (BovineSpongiform Encephalopathy) in cattle, and CWD (Chronic Wasting Disease)in deer.

As used herein, the term “prion contaminated surface” refers to asurface contaminated with (or potentially contaminated with) aninfective prion particle.

As used herein, “effective amount” is used to describe the amount of aparticular substance or combination of substances within a formulationdescribed herein to achieve a decrease in the titre of infective prionparticles.

As used herein, the term “peracid” is synonymous with peroxyacid,peroxycarboxylic acid, peroxy acid, percarboxylic acid and peroxoicacid.

As used herein, the term “peracetic acid” is abbreviated as “PAA” and issynonymous with peroxyacetic acid, ethaneperoxoic acid and all othersynonyms of CAS Registry Number 79-21-0 and will also include both theprotonated and unprotonated (i.e. peracetyl ions) forms.

As used herein, the term “perhydrolysis” is defined as the reaction of aselected substrate (an “activator”) with peroxide to form a peracid.Typically, inorganic peroxide is reacted with the activator to producethe peracid. In one embodiment, the peracid is peracetic acid.

As used herein, the term “biological contaminants” refers to one or moreunwanted and/or pathogenic biological entities including, but notlimited to microorganisms, spores, viruses, prions, and mixturesthereof. The present invention is particularly effective in destroyingand/or degrading prions.

As used herein, the term “disinfect” refers to the process ofdestruction of or prevention of the growth of biological contaminants.As used herein, the term “disinfectant” refers to a composition thatdisinfects by destroying, neutralizing, or inhibiting the growth ofbiological contaminants. Typically, disinfectants are used to treatinanimate objects or surfaces.

As used herein, the term “decontaminate” means to make safe byeliminating harmful substances (e.g. prions particles).

As used herein, the terms “peroxygen source”, “source of peroxygen”, and“oxygen source” refer to compounds capable of providing and effectiveamount of hydrogen peroxide including, but not limited to hydrogenperoxide, hydrogen peroxide adducts (e.g., urea-hydrogen peroxide adduct(carbamide peroxide)), perborates, and percarbonates. In a preferredembodiment, the peroxygen source is sodium perborate.

As used herein, the term “oxidizing agent system” refers a system (e.g.a composition or formulation) that provides an effective amount of anoxidizing agent. In one embodiment, the oxidizing agent system comprisesa peroxygen source and a peracid activator that (when mixed undersuitable aqueous reaction conditions) generates an efficacious amount ofperacid, preferably peracetic acid.

Aqueous Prion-Degrading Composition

The present invention relates to compositions for use in priondecontamination of prion-contaminated entities. In one aspect, theinvention provides a prion-degrading composition comprising an oxidizingagent, one or more proteases and a surfactant such as an ionicsurfactant/detergent.

Oxidizing Agent

The oxidizing agent may be any chemical entity or mixture capable ofoxidizing (or of catalysing the oxidation of) proteins, preferably prionprotein(s).

The oxidizing agent may be any of those known to be effective informulations with biocidal efficacy. The oxidizing agent may be selectedfrom the group consisting of peroxide, persalt, peracids, persulfates,peroxyphthalates, organic chlorines, chlorine dioxide, and stablemixtures thereof. Examples include sodium hypochlorite, sodiumperborate, sodium percarbonate, hydrogen peroxide, peracetic acid,chlorine dioxide, potassium peroxymonopersulfate, sodiumdichloroisocyanurate, trichlorocyanuric acid, and stable mixturesthereof.

In one embodiment, the oxidizing agent is provided as a liquid or asolid dissolved in a polar solvent (e.g., water). Once dissolved, theoxidizing agent provides a suitable concentration of active oxidizingagent species in the prion-degrading composition. “Active oxidizingagent” refers to total oxidizing species as determined by availableoxygen AVOX/iodometric titration, or other suitable method(thiosulfatimetric method, permanganometric method, cerimetric method),that is, oxidizing power (see “Peroxide Chemistry”, Waldemar Adam,Editor, Wiley-VCH, Weinheim, Germany, 2001 and “Organic Peroxides”,Daniel Swern, Editor, John Wiley and Sons, Inc., Hoboken, N.J., 1971).In the iodometric method, the oxidizing power of products isconveniently measured in terms of the amount of iodine liberated byreaction with potassium iodide (determined by a subsequent titration ofthat iodine). The procedure is standard in the art, and the results canbe expressed in terms of available hypohalite, peracid, of halogen, orof oxygen, or simply as “oxidizing power”.

Preferred oxidizing agents are those with good low temperature efficacyand good materials compatibility.

In a preferred embodiment, the oxidizing agent comprises peroxide, amodified peroxide, a peracid, such as peracetic acid, or mixturesthereof. This maybe an equilibrium mixture of peracetic acid, hydrogenperoxide and acetic acid or generated in situ from an appropriate sourceof peroxygen and an “activator”. Examples of activators (or peracidprecursors) include, but are not limited to tetraacetylethylenediamine(TAED), nonanoyloxybenzene sulfonate (NOBS), Sodium nonanoyloxybenzenesulfonate (SNOBS), nonyl amido caproyl oxybenzene sulfonate (NACA-OBS),carboxylic acid esters, triacetin, diacetin, acetic acid, tetracetylglycol uril (TAGU), diacetyl hexahydrotriazine oxide, N-acyl lactams,acetyl triethyl citrate (ACT), glucose pentaacetate, sucroseoctaacetate, and acetyl salicylic acid, to mention a few. In a preferredembodiment, the activator is an N-acyl donor, such as TAED, SNOBS, TAGU,N-acyl lactams. In a further preferred aspect, the activator is TAED.

It will be recognised that the peracetic acid and peroxide will exist inboth protonated and de-protonated forms, the ratio being dependent onthe pH and the pKa of the relevant species. Examples of commerciallyavailable peracid-based disinfectants include, but are not limited toPERASAFE™ Sterilant and RelyOn™ Disinfectant. PERASAFE™ Sterilant is apowdered blend of an oxygen source (40-60 wt % sodium perborate and10-30 wt % tetraacetylethylene diamine), stabilizer, corrosioninhibitor, buffer, and surfactant, that when mixed with water, generatesan oxidizing agent system comprising approximately 0.26% peracetic acid(as a mixture of peracetic acid and peracetyl ions) at pH 8.0, whenprepared according to manufacturer's instructions.

Preferably the oxidizing agent is a peroxygen system. A peroxygen systemis a combination of peroxygen source (e.g., a persalt such as sodiumperborate or sodium percarbonate) and an activator such as tetraacetylethylenediamine (TAED) or N-acetyl caprolactam, or other activator aslisted hereinabove, which generates peracetic acid and peracetic anionsin situ upon dissolution. Preferably, the peroxygen system is used inthe liquid phase, preferably in solution, preferably in aqueoussolution.

In a one aspect, the oxidizing agent comprises at least one C1 to C10aliphatic peracid. In a preferred aspect, the oxidizing agent comprisesperacetyl ions (CH₃C(O)OO⁻) and/or peracetic acid (CH₃C(O)OOH; CAS79-21-0). Preferably the oxidizing agent comprises hydrogen peroxideand/or peracetic acid, more preferably the oxidizing agent compriseshydrogen peroxide, peracetic acid and peracetyl ions. A preferredoxidizing agent is PERASAFE™ Sterilant as supplied by AntecInternational, Sudbury, UK (a subsidiary of E. I. DuPont de Nemours andCompany, Inc., Wilmington, Del., USA). In a particular embodiment,PERASAFE™ Sterilant is formulated for a stock solution ranging from 0.1×to 20× PERASAFE™ Sterilant, preferably from 0.5× to 5× PERASAFE™Sterilant, more preferably 0.5× to 2× PERASAFE™ Sterilant. In case ofconflict, preferably PERASAFE™ Sterilant is formulated in accordancewith the manufacturers' instructions, i.e., 81 g/5 litres for a 1×PERASAFE™ Sterilant stock solution.

The amount of oxidizing agent in the aqueous prion-degrading compositionis an effective amount, i.e., an amount that, when used in thecomposition with the other components is effective for priondegradation. Preferably, when the oxidizing agent is peracetic acid, itis used at a concentration equivalent to 0.26 w/v % peracetic acid(0.26% arises from use of PERASAFE™ Sterilant at 16.2 g/L so that 0.1%remains 24 hours after make up). It has been surprisingly discoveredthat there is synergy between the oxidizing agent and the protease(prion-degrading enzyme) used herein. Thus, reduced use of the oxidizingagent can be offset by increased use of enzyme and vice versa. The exactamounts of both oxidizing agent and protease can be determined by in-useconditions, cost considerations and the degree of efficacy required.These amounts can be readily determined by one skilled in the art.

Preferably the oxidizing agent is used at near-neutral pH. In oneembodiment, the pH range is about 5 to about 9, preferably about 6 toabout 8, and most preferably about 8 for reasons of materialscompatibility.

Optionally, hydrogen peroxide is also included in the oxidizingcomposition in addition to other oxidizing agent. Preferably hydrogenperoxide is in solution, more preferably in aqueous solution.

Optionally, acetic acid is also included in the oxidising composition.

Proteases

The aqueous prion-degrading composition comprises at least one proteasethat, when combined with the oxidizing agent, is present in an amounteffective for prion degradation.

Proteases or peptidases (used interchangeably) are enzymes which cleavethe amide linkages in protein substrates (see Walsh, Enzymatic ReactionMechanisms, W.H. Freeman and Company, San Francisco, Chapter 3 (1979)and Rao et al., Microbiol. Mol. Biol. Rev., 62(3):597-635 (1998)).Proteases includes any enzyme belonging to the EC 3.4 enzyme group, asdefined by the Nomenclature Committee of the International Union ofBiochemistry and Molecular Biology. These enzymes can be grosslysubdivided into two major categories, exopeptidases and endopeptidases,depending upon their site of action.

As proteases are ubiquitous to all living organisms, suitableprion-degrading proteases for use in the composition of this inventioncan be isolated from a variety of eukaryotic and/or prokaryoticorganisms. Commercially-useful proteases have been isolated from plants(e.g. papain, bromelain, and keratinases), animals (e.g., trypsin,pepsin, and rennin), and microbes such as fungi (e.g., Aspergillusoryzae proteases) and bacteria (especially well-known proteases isolatedfrom the genus Bacillus). Bacillus species secrete two extracellulartypes of protease, neutral proteases (many of which aremetallo-endoproteases, such as NEUTRASE®, typically active in a generalpH range of about 5 to about 8, see U.S. Pat. No. 6,636,526) andalkaline proteases (such as ALCALASE®, typically characterized by highactivity at alkaline pH; see GENBANK® P00780 and P00782; van der Ostenet al., J. Biotechnol., 28:55-68 (1993)).

A sub-group of the serine proteases tentatively designated subtilaseshas been proposed by Siezen, et al., supra. These serine proteases areuseful in the composition, method and kit of this invention. They aredefined by homology analysis of more than 40 amino acid sequences ofserine proteases previously referred to as subtilisin-like proteases. Asubtilisin was previously defined as a serine protease produced byGram-positive bacteria or fungi, and according to Siezen, et al., now isa subgroup of the subtilases (or “subtilisin proteases”). A wide varietyof subtilisins have been identified, and the amino acid sequence of anumber of subtilisins have been determined. These include more than sixsubtilisins from Bacillus strains, namely, subtilisin 168, subtilisinBPN', subtilisin Carlsberg, subtilisin Y, subtilisin amylosacchariticus,and mesentericopeptidase (Kurihara et al., J. Biol. Chem. 247 5629-5631(1972); Wells et al., Nucleic Acids Res. 11 7911-7925 (1983); Stahl andFerrari, J. Bacteriol. 159 811-819 (1984), Jacobs, et al., Nucl. AcidsRes. 13 8913-8926 (1985); Nedkov et al., Biol. Chem. Hoppe-Seyler 366421- 430 (1985), Svendsen, et al., FEBS Lett. 196 228-232 (1986)), onesubtilisin from an actinomycetales, thermitase from Thermoactinomycesvulgaris (Meloun et al., FEBS Lett. 198 195-200 (1985)), and one fungalsubtilisin, Proteinase K from Tritirachium album (Jany and Mayer, Biol.Chem. Hoppe-Seyler 366 584-492 (1985)). For further reference, see TableI from Siezen, et al., supra.

Subtilisins are well-characterized physically and chemically. Inaddition to knowledge of the primary structure (amino acid sequence) ofthese enzymes, over 50 high resolution X-ray structures of subtilisinshave been determined which delineate the binding of substrate,transition state, products, at least three different proteaseinhibitors, and define the structural consequences for natural variation(Kraut, Ann. Rev. Biochem. 46 331-358 (1977)).

One subgroup of particularly useful subtilases for this invention, I-S1,comprises the “classical” subtilisins, such as subtilisin 168,subtilisin BPN', subtilisin Carlsberg (e.g., ALCALASE®, available fromNovozymes A/S, Bagsvaerd, Denmark), and subtilisin DY.

A further subgroup of the subtilases I-S2, is recognized by Siezen, etal. (supra). Sub-group I-S2 proteases are also useful proteases in thisinvention and are described as highly alkaline subtilisins and compriseenzymes such as subtilisin PB92 (e.g., MAXACAL®, Gist-Brocades NV,Denmark), subtilisin 309 (e.g., SAVINASE®, Novozymes), subtilisin 147(e.g., ESPERASE®, Novozymes), and alkaline elastase YaB.

Random and site-directed mutations of the subtilase gene have botharisen from knowledge of the physical and chemical properties of theenzyme and contributed information relating to subtilase's catalyticactivity, substrate specificity, tertiary structure, etc. (Wells, etal., Proc. Natl. Acad. Sci. U.S.A. 84:1219-1223 (1987); Wells, et al.,Phil. Trans. R. Soc. Lond.A. 317:415-423 (1986); Hwang and Warshel,Biochem. 26 2669-2673 (1987); Rao, et al., Nature 328:551-554 (1987);Carter, et al., Proteins 6:240-248 (1989); Graycar, et al., Annals ofthe New York Academy of Sciences 672 71-79 (1992); and Takagi, Int. J.Biochem. 25 307-312 (1993).

Examples of proteases and protease variants which are useful in thisinvention have been disclosed in numerous United States patents andpatent applications including, but not limited to U.S. patentapplication publications U.S. 200502391885 A1 and U.S. 20060147499 A1and issued U.S. Patents: U.S. Pat. No. 5,500,364; U.S. Pat No.6,506,589; U.S. Pat. No. 6,555,355; U.S. Pat. No. 6,558,938; U.S. Pat.No. 6,558,939; U.S. Pat. No. 6,605,458; U.S. Pat. No. 6,632,646; U.S.Pat. No. 6,682,924; U.S. Pat. No. 6,773,907; U.S. Pat. No. 6,777,218;U.S. Pat. No. 6,780,629; U.S. Pat. No. 6,808,913; U.S. Pat. No.6,835,821; U.S. Pat. No. 6,893,855; U.S. Pat. No. 6,921,657; U.S. Pat.No. 7,026,53; U.S. Pat. No. 7,098,017; and U.S. Pat. No. 7,109,016.

Examples of commercially available proteases include, but are notlimited to the group consisting of Bacillus sp. (e.g. Bacillus subtilis,Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus lentus,etc.), proteases such as subtilisins (ALCALASE®; available fromNovozymes A/S, Bagsvaerd, Denmark), the neutral protease NEUTRASE®(available from Novozymes), EVERLASE® (Novozymes; a protease fromBacillus sp. available from Sigma-Aldrich, catalog #P5985), POLARZYME®(Novozymes; a protease engineered for low temperature washingapplications), SAVINASE® (available from Novozymes), Sus scrofa pepsin,Carica papaya chymopapain, Ananas comosus bromelain, Carica papayapapain, Streptomyces griseus PRONASE® (also known as PRONASE E® orPRONASE®), Tritirachium album Proteinase K (includingrecombinantly-produced Proteinase K from Pichia pastoris), and mixturesthereof. These proteases are commercially available enzymes and may beavailable in a variety of product forms including powders, tablets, andliquid formulations. In another aspect, the commercially-availableenzymes(s) may be optionally purified or partially purified prior to usein the present formulations. Means to purify proteins are well-known inthe art. In a preferred embodiment, the protease is selected from thegroup consisting of Proteinase K, PRONASE®, ALCALASE®, NEUTRASE®, andmixtures thereof.

In one aspect, the composition of this invention comprises a combinationof two or more proteases. Preferably when two or more proteases arepresent in the composition, the proteases are selected from the groupconsisting of PRONASE®, proteinase K, ALCALASE®, and NEUTRASE®.

In a preferred embodiment, the protease is a combination of Proteinase Kand PRONASE®, ALCALASE® and NEUTRASE® or ALCALASE® and PRONASE®. Whenthe composition comprises two or more proteases, the proteases can beused sequentially or simultaneously in a mixture for proteolysis.However, it should be noted that when contacting a prion-contaminatedentity with several proteases at once, individual activities can bereduced and compensation might be necessary e.g., by longer time ofcontact. This is discussed in more detail below.

As is plain to a person of ordinary skill in the art, the higher theconcentration of protease(s), the greater and more rapid destruction isachieved. Combinations of protease concentration and time may be chosenaccording to need. These can be optimised by routine trial and error.

Proteases are susceptible to genetic and/or peptide or chemical levelmanipulation or modification. It will be apparent to a person skilled inthe art that truncations, mutations or adaptation of the proteases(e.g., to make them more protease resistant themselves) does notinterfere with the invention provided that the peptidase activity of theenzyme(s) is retained by such manipulation(s). (Indeed, it is acceptedthat PRONASE® is more in the nature of a fractionated proteasepreparation rather than a recombinantly purified enzyme, and use of asub-fractionation product of PRONASE® or of a cloned and recombinantlypurified fraction of the peptidase(s) comprised by PRONASE® are embracedby the present invention). Thermostable proteases are particularlypreferred, whether obtained by modification of existing proteases or bycloning proteases from thermophilic organisms.

In general, total protease, added to the composition of the invention isabout 1.6 g/l (1.6 mg/mL). However, an effective amount of protease canand will vary, for example, from less than 0.1 to over 20 g/l, dependingon the amount of oxidizing agent present. Typical ranges are from 0.1 to5 g/l or from 1 to 4 g/l. More protease will be added if less oxidizingagent is present and conversely, less protease may be used in aneffective composition if more oxidizing agent is present.

Surfactant/Detergent

The prion-degrading composition of the present invention comprises asurfactant. As used herein, the terms “surfactant” and “detergent” willbe used interchangeably and include zwitterionic surfactants, anionicsurfactants, cationic surfactants, and non-ionic surfactants. Examplesof surfactants include, but are not limited to anionic surfactants suchas sodium dodecyl sulfate (SDS), ammonium lauryl sulfate and other alkylsulfate salts, sodium 1-octanesulfonate monohydrate, N-lauroylsarcosinesodium salt, sodium lauryl sulfate, sodium lauryl ether sulfate (SLES),sodium taurodeoxycholate hydrate, and alkyl benzene sulfonate; cationicsurfactants such as cetyl trimethylammonium bromide and otheralkyltrimethylammonium salts, cetylpyridinium chloride, polyethoxylatedtallow amine, benzalkonium chloride,and benzethonium chloride;zwitterionic (amphoteric) surfactants such as dodecyl betaine, dodecyldimethylamine oxide, cocamidopropyl betaine, and coco ampho glycinate;and non-ionic surfactants such as alkyl poly(ethylene oxide), copolymersof poly(ethylene oxide) and poly(propylene oxide, alkyl polyglucosides(e.g., acetyl glucoside), decyl maltoside, fatty alcohols, cetylalcohol, oleyl alcohol, cocamide monoethanolamine, cocamidediethanolamine, and cocamide triethanolamine).

In a particular embodiment the surfactant is selected from the groupconsisting of sodium dodecylbenzene sulfonate, lauryl ether sulfate,ethylene oxide/propylene oxide alkyl phenol condensate, polyglycol etherof fatty alcohols, fatty acid ethylene oxide condensate, polyglycolether of alkyl phenols, fatty alcohol ethoxylate, sodium lauryl ethersulfate, sodium dodecyl sulfate, and combinations of two or morethereof.

In another particular embodiment, the surfactant is a cationic oranionic surfactant, preferably an anionic surfactant, and morepreferably sodium dodecyl sulfate (SDS), sodium taurodeoxycholatehydrate, sodium 1-octanesulfonate monohydrate, lithium dodecyl sulphate,N-lauroylsarcosine sodium salt, or a combination of two or more thereof.Preferably the surfactant is SDS.

The surfactant can be used at any effective concentration. This may beeasily determined and/or optimised by routine trial and error. When thesurfactant is SDS, the final concentration of the surfactant with regardto the contacting a prion-contaminated entity with a surfactant ispreferably about 1% (weight percent) to about 6%, more preferably about1% to about 3%, even more preferably about 1%, and most preferably 1%,based on Western Blot optimization. Again, there is synergy between theenzymes, surfactant and oxidizing agent and the component levels can bevaried to fit use requirements.

Proteases can be adversely affected (e.g., suffer reduced activity orloss of activity) in the presence of excess surfactant. Individualproteases have individual characteristics, and it is well within theabilities of a person skilled in the art to avoid loss of activity dueto surfactant action. Manufacturer' guidance should be followed whereverpossible. Advantageously surfactant level(s) are limited so as not tosignificantly inhibit protease activity for prion-degradation before andat the time of contact with protease.

Additional Components

In a preferred embodiment, the prion-degrading composition comprises atleast one corrosion inhibitor. Although the corrosion inhibitor may notcontribute to the efficacy of the prion-degrading composition, use of atleast one corrosion inhibitor is preferred, especially whendecontaminating delicate instruments. Examples of corrosion inhibitorsare described in U.S. Pat. No. 5,077,008 and typically include, but arenot limited to triazoles (e.g., benzotriazole), azoles, phosphates, andbenzoates. In one embodiment the corrosion inhibitor is benzotriazole ortolyltriazole. In a preferred embodiment, the corrosion inhibitor isbenzotriazole.

In another preferred embodiment, the prion-degrading compositioncomprises at least one peroxygen stabilizer. Peroxygen stabilizers areknown, see for example, U.S. Pat. No. 5,624,634. Examples of peroxygenstabilizers suitable for use in the composition of this inventioninclude hydrogen peroxide stabilizers and peracid stabilizers. Suchstabilizers include, but are not limited to, amino tri(methylenephosphonic acid) (DEQUEST® 2000 series),1-hydroxyethylidene-1,1-diphosphonic acid (HEDP; DEQUEST® 2010 series),hexamethylene diamine tetramethylene phosphonic acid (DEQUEST® 2050series), bis hexamethylene triamine penta methylene phosphonic acid(DEQUEST® 2090 series, diethylenetriamine pentamethylene phosphonate(DEQUEST®) 2060 series), ethylene diamine tetramethylene phosphonic acid(DEQUEST® 2041), dipicolinic acid, phosphonic acids and salts thereof.DEQUEST stabilizers are available from Solutia, Inc., St. Louis, Mo. Ina preferred embodiment, the peroxygen stabilizer is ethylene diaminetetramethylene phosphonic acid (DEQUEST® 2041) or1-hydroxyethylidene-1,1-diphosphonic acid (HEDP, DEQUEST® 2010) and/orsalts thereof.

Preferred Embodiments

In one preferred embodiment the prion-degrading composition comprises atleast two proteases. In one embodiment, the prion-degrading compositioncomprises at least two proteases and the surfactant is sodium dodecylsulfate. In a further preferred embodiment, the two proteases areselected from the group consisting of PRONASE®, ALCALASE®, NEUTRASE®,and Proteinase K and the surfactant is sodium dodecyl sulfate. Morepreferably, the prion-degrading composition comprises two proteases andthe two proteases are NEUTRASE® and ALCALASE®.

Preferably the oxidizing agent of the prion-degrading compositioncomprises peracetic acid at pH from about 6 to about 8, and henceconsists of both protonated and deprotonated forms.

Method for Decontaminating Prion-Infected Entity

The present invention provides a method for decontaminating aprion-contaminated entity which comprises contacting the contaminatedentity with (a) an effective amount of at least one oxidizing agent, (b)an effective amount of at least one protease, and (c) and effectiveamount of at least one surfactant. The contacting step is performed fora period of time sufficient to degrade the prion particles. Preferredoxidizing agents, proteases, surfactants and their amounts for use inthe methods of this invention are described hereinabove.

The method optionally comprises the step of autoclaving the entity aftercontacting with the prion-degrading composition. The protocol for use ofthe prion-degrading composition is compatible with existing hardwaresuch as machines used for pre-washing medical instruments prior toautoclaving.

In one embodiment, at least one oxidizing agent and at least twoprotease steps are used in combination with a surfactant.

In another aspect the invention relates to a method as described abovewherein the first protease is NEUTRASE® and the second protease isALCALASE®.

Simultaneous/Sequential Contacting

The contacting steps of the method of this invention can be performedsequentially or simultaneously. Preferably, the prion-contaminatedentity is contacted with the at least one protease either simultaneouslywith or following contact with the oxidizing agent.

Where more than one protease is used, the proteases may be combined intoa single step. However, protease activity can be lowered in such anembodiment due to each protease digesting the other. Still, theindividual steps in the methods of the present invention may be carriedout sequentially or simultaneously. The steps of the methods of thisinvention may be repeated and any of the methods may further comprisewashing steps to remove the prion-degrading composition, e.g., from asolid surface.

When the contacting steps are performed sequentially, the method of thisinvention comprises (a) first contacting the prion-contaminated entitywith a surfactant, and then (b) contacting the entity with an oxidizingagent, and then (c) contacting the entity with at least one protease.

In another embodiment, a sequential method for prion decontamination ordisinfection of a prion-contaminated entity comprises: (a) firstcontacting the entity with a surfactant, and then (b) contacting theentity with an oxidizing agent, and then (c) contacting the entity witha first protease, and (d) contacting the entity with a second protease.When contacting the prion-contaminated entity with two proteases, it maybe advantageous to remove all or a portion of the first protease beforethe entity is contacted with the second protease. In one embodiment,substantially all of the first protease is removed before contact withthe second protease. This applies equally to each protease step in amulti-step sequence.

In one embodiment, it may be further desirable to separate thesurfactant application step from the protease application step(s) if thesurfactant adversely affects the protease activity. In this embodiment,at least a proportion of the surfactant is removed (or diluted) beforethe entity is contacted with a protease in order to maximize proteaseactivity.

In a preferred embodiment, an aqueous prion-degrading compositioncomprising an effective amount of oxidizing agent, an effective amountof at least two proteases, and an effective amount of at least onesurfactant, are combined and simultaneously contacted with aprion-contaminated entity, i.e., in a single step. Thus, the surfactantis mixed together with the protease and the oxidizing agent, preferablyimmediately prior to use so that a single aqueous composition is appliedto the prion-contaminated entity. In this embodiment, a lowertemperature (see below) is typically used to avoid inactivation of theenzymes (proteases).

In a further preferred embodiment, all of the components of theprion-degrading composition are mixed together in water to form a singleaqueous prion-degrading composition which is subsequently contacted witha prion-contaminated entity.

Alternatively, in one embodiment, the surfactant is contacted with theprion-contaminated entity prior to contacting the entity simultaneouslywith the oxidizing agent and the proteases.

In a preferred aspect the invention provides a method for priondecontamination comprising simultaneously contacting aprion-contaminated entity with a surfactant, an oxidizing agent,NEUTRASE® and ALCALASE® to provide a reaction mixture. Preferably thesurfactant is SDS. Optionally, the method further comprises, after thecontacting step, autoclaving the reaction mixture.

Whether sequential or simultaneous method is employed, the reaction timeor incubation time for each step will typically be less than 24 hours,preferably less than 2 hours, more preferably less than 1 hour, and mostpreferably less than 20 minutes.

Autoclaving

In one aspect the invention, the method of this invention furthercomprises, autoclaving the entity.

Autoclaving, if employed as described herein, can be carried outfollowing any suitable autoclave cycle. Typical cycles include 121° C.for 18 minutes or preferably 134° C. for 18 minutes. Alternative cyclesmay be chosen by the operator to suit their particular needs. Extendedautoclave cycles may be advantageously employed. Autoclaving in watercan enhance the prion destructive effects and is therefore preferredwhere autoclaving is used.

Advantageously an autoclaving step is performed as a final step in themethods of the present invention, that is, autoclaving is performedafter contacting step(s). An autoclaving step provides the advantage ofminimizing spread of infection via the prion-contaminated entity, and isparticularly advantageous when the entity is a surgical instrument.Furthermore, by combining contacting step(s) with autoclaving in thismanner, there may advantageously be a multiplicative increase inefficacy, i.e., if each method can reduce infectious titre by 5 logsthen combining them may reduce infectivity by even more, such as by 10logs.

Preferably autoclaving is avoided or performed for reduced time such asless than 3 hours, preferably autoclaving is omitted; especially forfragile medical instruments.

Temperature

The prion-degrading composition and its individual components, such as,in a sequential method, can be contacted with the prion-contaminatedentity over a range of temperatures. In one embodiment, the contactingstep(s) is at a temperature in the range of 15° C. to about 80° C.,preferably about 20° C. to about 60° C., more preferably about 40° C. toabout 60° C., and most preferably about 45° C. to about 55° C.

When contacting the surfactant in a separate step that does not includea protease, the surfactant may be contacted with the prion-contaminatedentity at any suitable temperature. Indeed, a high temperature may beused. A separate step for contacting surfactant with the entity isflexible and is preferably performed at a temperature of at least 70°C., preferably at least 80° C., preferably at least 90° C., preferablyat least 100° C.

The temperature may be constrained by the nature of the entity, forexample some medical equipment such as endoscopes cannot tolerate hightemperatures such as those used in autoclaving. For these situations,the methods of the invention advantageously do not involve autoclaveconditions, and the temperature choice should be made by the operatorwith regard to the tolerances of the entity being decontaminated.Examples of methods according to the present invention which avoid theuse of autoclave conditions may be found in the Examples section.Advantageously methods according to the present invention such as thosein the Examples may replace conventional prior art treatments such asLpH®, LpH®se, and EndozymePlus treatment. See, U.S. Patent ApplicationPublication No. 2006/0217282 A1.

Whether sequential or simultaneous method is employed, the reaction timeor incubation time for each step will typically be less than 24 hours,preferably less than 2 hours, more preferably less than 1 hour, and mostpreferably less than 20 minutes.

Incubation temperatures, that is, temperatures at which the protease(s)are contacted with the entity will vary according to the protease used.Generally, any temperature from room temperature (e.g., 15-20° C.) up toabout 80° C. is acceptable, wherein about 20° C. to about 60° C. is morepreferred, wherein about 40° C. to about 60° C. is even more preferred,and about 45° C. to about 55° C. is most preferred. As the temperaturemoves away from the optimum for a particular protease, deactivation ofthe prion contaminants takes longer. Clearly, this can be compensatedfor by incubating for a longer time or using a greater concentration ofprotease. At temperatures above 60° C. activities can be lower and theenzymes can become inactivated, but clearly individual proteasepreparations will have individual deactivation temperatures and themanufacturers' guidance should be followed wherever possible.

As used herein, “low temperature” means less than 134° C., preferablyless than 121° C., more preferably less than 100° C., even morepreferably less than 90° C., even more preferably less than 80° C., evenmore preferably less than 70° C., yet even more preferably less than 60°C., and most preferably means a temperature such as about 35° C. toabout 60° C.

Miscellaneous Method Features

Preferably, the contacting step(s) is carried out with agitation.Preferably said agitation is a rotary agitation, e.g., at about 750 rpm.

Examples presented herein include conditions optimal for use inautomated washing machines. Furthermore, the conditions chosen areadvantageously low in cost.

Advantageously, when the prion-contaminated entity is a medicalinstrument, the methods for prion decontamination are performed in amedical instrument sterilizer.

Prion-Contaminated Entity

The prion-contaminated entity may be any physical item for which it isdesired to deactivate and/or remove prions. The term embraces fluids aswell as solids (objects) such as devices or medical instruments(including surgical instruments). Fluids include biological fluids. Theprions to be deactivated or removed may be in the entity (e.g., insolution or suspension in a fluid), or may be on the entity (e.g.,bound, attached or otherwise associated with a surface of a solidentity). Thus, the entity may have a surface. Entities having surfacesinclude, for example, a medical instrument, a laboratory instrument, aclean-room surface, a countertop, or the surface of an area or equipmentused for food preparation or processing or the surface of an enclosureused to house animals. The surface may comprise metal, plastic or anyother relevant material of construction. The metal may be steel such assurgical steel.

When the prion-contaminated entity is a solid surface, it may also belaboratory countertops, laboratory instruments and laboratory equipment,for example in a clean room for research, production and testing ofpharmaceutical and biological compounds.

Non-limiting examples of entities, which may be contaminated with prionsand which can be advantageously and effectively decontaminated accordingto the methods of this invention, include surgical equipment surfacesfrom veterinary or hospital settings as wells as surfaces that come incontact with said surgical equipment. Preferably the entity is a medicalinstrument, including fragile instruments, such as a laparoscope orendoscope.

The entity may be used in the food processing industry. Thus,prion-contaminated entity may be selected from the group consisting oftanks, conveyors, floors, drains, coolers, freezers, equipment surfaces,walls, valves, belts, pipes, drains, joints, crevasses, and combinationsof two or more thereof. The prion-contaminated entity may be selectedfrom a surface of a barn or stable for livestock, such as poultry,cattle, dairy cows, goats, horses and pigs; and or hatcheries andhatcheries for poultry or shrimp.

The target surface may be contacted with the presentprion-decontaminating compositions using any number of means. The time,temperature, and effective concentration used when contacting thedesired locus can be easily determined by one of skill in the art.Specific contacting methods include spraying, treating, immersing,flushing, pouring on or in, mixing, combining, painting, coating,applying, affixing to and otherwise communicating the presentprion-decontaminating composition(s) with the surface to be treated,i.e., known or suspected of being contaminated with prion particles.

Decontamination

Decontamination refers to reduction in prion titre in a specific sampleor setting. Decontamination may refer to the removal of prions from asurface whether or not said prions are deactivated. Thus,decontamination includes deactivation and also includes the eliminationof prions without regard to whether or not they aredestroyed/deactivated. When decontaminating, it is important that prioninfectivity is removed from the surface or solution beingdecontaminated. This may be by destruction (deactivation) or by simpleseparation. Thus, the methods of this invention may further comprise,after contacting the prion-contaminated entity with (a) an effectiveamount of at least one oxidizing agent, (b) an effective amount of atleast one protease, and (c) and effective amount of at least onesurfactant, separating the treated entity from the treating reagents.The important aspect is that prions (i.e., PrP^(Sc)) are no longerassociated with the surface or solution being decontaminated or arereduced in number and/or titre. Clearly, if non-infective prionfragments remain adhered to a treated surface after decontamination,this would not materially affect the decontamination or the fact thatthe surface had been successfully decontaminated.

In one aspect of this invention, the prion-degrading compositions andmethods are used to decrease and/or eliminate iatrogenic CJD.

Decontamination may be assessed by any suitable assay. Preferably, theassay used is Western Blotting or bioassay. Clearly assays such asbioassays and/or Western Blotting assays have a sensitivity limit. Solong as prion titre (prion number/infectivity) has been reduced, thenprion decontamination will be considered to have taken place.

Preferably prion decontamination is 100 fold, preferably 1000 fold,preferably 10,000 fold, preferably 100,000 fold, preferably 1,000,000fold or even more. Preferably prions are completely eliminated ordeactivated.

Disinfection refers to cleansing so as to destroy or prevent the growthof pathogenic microorganisms in addition to the decontaminationassociated with prion infectivity. The disinfection occurs by thecombined action of an effective amount of oxidizing agent (e.g.,peracetic acid), an effective amount of the protease(s), and aneffective amount of the surfactant.

Assay Methods

The reduction in prions produced by the methods of the present inventionmay be monitored by any suitable means known in the art. Specificexamples of suitable assay techniques are provided herein to illustratethe assessment of prion reduction.

Clearly, certain methods will present themselves as more suitable for agiven situation than other methods. For example, if priondecontamination is taking place in solution, then a Western Blottingapproach might be most suitable. If prion decontamination is takingplace on a surface, then direct visualisation on that surface might bemost suitable. Alternatively for prion decontamination taking place on asurface, bioassay might be the most suitable. Choice of individual assaymethods for individual situations is well within the capabilities of aperson skilled in the art. It will be appreciated that in manysituations the most important indicator is loss/reduction ofinfectivity. Currently, prion infectivity is most usually assessed bybioassay. However, biochemical assay of the infective conformer PrP^(Sc)is equally appropriate.

An example of a suitable monitoring method is an immunoblotting assay.Advantageously the immunoblotting assay is, or is based on, the assaydescribed in Wadsworth et al. (Lancet 358:171-180 (2001)).

An example of a suitable monitoring method is a bioassay. Bioassaymethods are generally geared towards the individual prion species beingassayed. Selection of suitable bioassay methods is advantageously basedon the prion species being assayed.

Kits

The present invention also relates to kits for use in decontamination ofprion-contaminated entities. In one embodiment, the kit comprises a setof reactants wherein a first reactant comprises an oxidizing agent, asecond reactant comprises at least one protease and a third reactantcomprises a surfactant.

In a preferred embodiment the second reactant comprises at least twoproteases. In a more preferred embodiment, the at least one or the atleast two proteases are selected from the group consisting of PRONASE®,Proteinase K, ALCALASE®, and NEUTRASE®. In a more preferred embodiment,the second reactant comprises at least two proteases, wherein the twoproteases are ALCALASE®, and NEUTRASE®.

In a further embodiment, the kit comprises one or more additionalreactants wherein the additional reactants are selected from the groupconsisting of pH adjusters, buffering agents, chelating agents,corrosion inhibitors, peroxygen stabilizers and mixtures thereof.Alternatively, the first, second or third reactant may further compriseone or more of the additional reactants.

In a further embodiment, each of the reactants in the kit are suppliedin solid form, preferably as powders, to promote storage stability. Thereactants of the kit are mixed with a polar solvent at an effectiveconcentration of each of the reactants. Once effectively mixed in thepolar solvent there is provided a prion-decontaminating compositionready to use. The preferred polar solvent is water.

The reagents used are water soluble, stable, and of low toxicity. Theprotocol for their use is compatible with existing hardware for exampleas used in hospital decontamination departments for pre-washing andautoclaving instruments. Thus the invention provides for decontaminationof prion infectivity from surgical instruments. Advantageously themethods of the present invention can be implemented using existingmachinery.

The invention is now illustrated by way of examples which should not beregarded as limiting in scope. In particular the step(s) in the Exampleswherein an entity is treated with a surfactant at high temperature is(are) not to be regarded as essential features of the invention, but is(are) merely advantageous optional steps.

Various modifications and variations of the described methods andcompositions of the present invention will be apparent to those skilledin the art without departing from the scope and spirit of the presentinvention. Although the present invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention which are obvious to those skilledin biochemistry and biotechnology or related fields are intended to bewithin the scope of the following claims.

EXAMPLES Example 1 Combined Oxidation and Protease Treatment

This example describes methods by which an entity contaminated with theinfectious material PrP^(Sc) can be decontaminated and the infectiousmaterial deactivated in an aqueous suspension by sequential exposure ofthe entity (in the example the entity is infected brain tissue) to anoxidizing agent plus SDS as surfactant and two proteolytic enzymes(ALCALASE® and NEUTRASE®).

Preparation of Tissue Samples:

Brain of CJD-infected human frontal cortex was homogenized to 20% (w/v)in PBS Dulbecco GIBCO-BRL 14190-094 (Paisley, UK), phosphate buffersolution, referred to hereinafter as “PBS”, by passing the brain tissuethrough 18-gauge, 21-gauge and 23-gauge needles to produce “homogenate”.The homogenate was diluted to 15% w/v with PBS, frozen in small aliquotsand stored at −70° C.

Protocal A: Use of ALCALASE®/NEUTRASE® Concentrations in the Presence ofPERASAFE™ Sterilant

Each reaction was performed at a total volume of 100 μL, containing 60μL of 10% w/v brain homogenate.

(a) A sample of brain homogenate, 60 μL of 10% w/v brain homogenate, wascontacted with 20 μL of 5× oxidizing agent PERASAFE™ Sterilant, preparedaccording to manufacturer's instructions for a 0.26% peracetic acidconcentration at pH 8.0 using phosphate buffered saline solution (PBS),and 5 μL of 20% w/v SDS. The resulting reaction mixture containedPERASAFE™ Sterilant and 1% w/v SDS.

(b) The reaction mixture was incubated at 50° C. for 10 minutes and 40μL of the reaction mixture was analysed.

(c) Treatment with a first protease: ALCALASE® (Novozymes). A solutionof 10 mg/mL ALCALASE® was prepared in water. A 3 μL aliquot of thisenzyme solution was added to the 85 μL solution of treated homogenateproduced by step (b) above. The final concentration of ALCALASE® was 300μg/mL.

(e) Treatment with a second protease: NEUTRASE® (Novozymes). A solutionof 10 mg/mL NEUTRASE® was prepared in water. A 12 μL aliquot of thisenzyme solution was added to the 88 μL solution of SDS/PERASAFE™Sterilant and ALCALASE®-treated homogenate produced in step (c) above.The final concentration of NEUTRASE® was 1.2 mg/mL. The mixture wasincubated at 50° C. for 10 minutes.

Detection of PrP^(Sc) by Western Blot

The materials from the SDS/PERASAFE™ Sterilant/ALCALASE®/NEUTRASE®(Protocol A) treatment described above were submitted to Western blotanalysis. The blots were visualized using the antibodies ICSM 18 andICSM 35 to detect any remaining PrP^(Sc) in the samples. Using eitherantibody, there was no detectable PrP^(Sc). For antibody information anddetection methods throughout, see EP934531 A1 and EP1565213. Allantibodies described herein can be found by reference to (PCT) PatentApplication No PCT/GB99/03617; UK Patent No. GB 2,348,203; AustralianPatent No AU 773763; South African Patent No. ZA 2001/3947; and U.S.Pat. No. 6,534,036.

It is clear from this example that the methods of the present inventionlead to significant prion decontamination. After treatment according toProtocol A, with oxidizing agent and two proteases, high levels ofdestruction of PrP were observed at all concentrations of protease asevidenced by a total lack of immunoreactivity, which is equivalent to atleast a 1200 fold reduction of infectivity. This estimate is based upona previously determined detection limit for the specific WesternBlotting protocol used (Wadsworth et al., Lancet 358(9277):171-180(2001)) which is a preferred assay method.

Using this assay method, it was determined that one can readily detect12 μL of a 10% w/v brain homogenate in the untreated control. Thesensitivity of western blotting for PrP is always 10 nl of 10% w/vhomogenate. Thus, if 12 μl of 10% w/v homogenate is the starting amountin an experiment and no signal is detectable by Western Blot then therehas been a greater than or equal to 1200 fold reduction in PrP.

Example 2 Prion Decontamination Comparison Using PERASAFE™ SterilantTreatment Only and PERASAFE™ Sterilant and Protease Treatment in aSingle Step

In this Example, prion decontamination is demonstrated according to thepresent invention by oxidation using PERASAFE™ Sterilant and proteasetreatment (Protocol C), compared with ineffective prior art treatment ofPERASAFE™ Sterilant only (Protocol B).

Protocol B: PERASAFE™ Sterilant Only Treatment (Comparative)

The following protocol was used to prepare samples treated only withPERASAFE™ Sterilant.

(a) A 5× PERASAFE™ Sterilant stock solution was prepared, 1 mL, byadding 81 mg of PERASAFE™ Sterilant powder to 1 mL of distilled anddeionized (dd) H₂O at 37° C. Vortex mixing was used to dissolve thepowder and the solution was incubated at 37° C. for 15 minutes.

(b) 300 μL of CJD 10% w/v brain homogenate was prepared and subjected tovortex mixing to ensure even suspension by spinning in a microfuge atminimum speed setting of 1 (80×g) for 1 minute.

(c) 200 μL of supernatant of the brain homogenate was removed andtransferred to a clean tube, which had been warmed to 37° C. 65 μL of 5×PERASAFE™ Sterilant stock solution was added and mixed with thehomogenate by vortex mixing.

(d) 65 μL of PBS was added to the mixture produced in step (c) mixedunder vortex.

(e) A 20 μL aliquot of homogenate mixture from step (d) was removed to aseparate tube containing 20 μL of 2× SDS loading buffer and then theseparate tube was frozen at −70° C. in approximately 5 minutes.

(f) The mixture remaining from step (d) was incubated at 37° C. withagitation (750 rpm).

(g) Further 20 μL aliquots were removed from the mixture produced instep (d) at times 5, 10, 30, 60 and 120 minutes to separate tubes eachcontaining 20 μL of 2× SDS loading buffer. Each aliquot was frozen at−70° C. in approximately 5 minutes.

(h) All aliquots were thawed simultaneously in a heating block at 100°C. Vortex mixing of the aliquots was timed for every two minutes for atotal of 10 minutes and the aliquots were centrifuged at maximum speedin a microfuge (15,000×g).

(i) For each 20 μL aliquot sample, the sample was loaded into a separatewell containing 16% acrylamide gel buffered with tris-glycine. The gelwas run at 200 volts for 80 minutes. The remaining 20 μL of sample wasfrozen and stored at −70° C.

(j) The acrylamide-gel treated samples from step (i) were subjected toWestern blot analysis onto polyvinylidene fluoride (PVDF) membrane(Imobilon-P) for either 90 minutes at 40 volts or at 15 volts overnight(greater than 12 hours).

(k) The membrane was blocked in 5% milk protein and PBS for 45-60minutes.

(l) The treated samples were incubated with primary antibody for 1 hour,ICSM35B at 0.2 μg/mL in PBS. 25 mL volume was used for a singlemembrane.

(m) Blots were then developed according to the Storm® system for gel andblot analysis and blot imaging system (Phosphorimager®) systemtechnology, available from Molecular Dynamics, Inc., Sunnyvale, Calif.),for quantification.

Protocol C: Proteolytic Degradation with Proteinase K and PRONASE® inConjunction with PERASAFE™ Sterilant Oxidation

The following protocol was used to prepare samples treated withPERASAFE™ Sterilant in conjunction with Proteinase K (PK) and PRONASE®proteases.

(a) A 5× PERASAFE™ Sterilant stock solution was prepared, 1 mL, byadding 81 mg of PERASAFE™ Sterilant powder to 1 mL of distilled anddeionized (dd) H₂O at 37° C. Vortex mixing was used to dissolve thepowder and the solution was incubated at 37° C. for 15 minutes.

(b) 300 μL of CJD 10% w/v brain homogenate was prepared and subjected tovortex mixing to ensure even suspension by spinning in a microfuge atminimum speed setting of 1 (80×g) for 1 minute.

(c) 200 μL of supernatant of the brain homogenate was removed andtransferred to a clean tube, which had been warmed to 37° C. 65 μL of 5×PERASAFE™ Sterilant stock solution was added and mixed with thehomogenate by vortex mixing.

(d) 55 μL of a 20% w/v SDS solution in dd H₂O was added to the mixtureproduced in step (c) and the resulting mixture was mixed under vortex.

(e) 10 μL of a 10 mg/ml solution of PK in dd H₂O and 10 μL of a 40 mg/mLsolution of PRONASE® in dd H₂O were added to the mixture produced instep (d) and the resulting mixture was mixed under vortex.

(f) A 20 μL aliquot of homogenate mixture from step (e) was removed to aseparate tube containing 20 μL of 2×SDS loading buffer and then theseparate tube was frozen at −70° C. in approximately 5 minutes.

(g) The mixture remaining from step (e) was incubated at 37° C. withagitation (750 rpm).

(h) Further 20 μL aliquots were removed from the mixture produced instep (e) at times 5, 10, 30, 60 and 120 minutes to separate tubes eachcontaining 20 μL of 2×SDS loading buffer. Each aliquot was frozen at−70° C. in approximately 5 minutes.

(i) All aliquots were thawed simultaneously in a heating block at 100°C. Vortex mixing of the aliquots was timed for every two minutes for atotal of 10 minutes and the aliquots were centrifuged at maximum speedin a microfuge (15,000×g).

(j) For each 20 μL aliquot sample, the sample was loaded into a separatewell containing 16% acrylamide gel buffered with tris-glycine. The gelwas run at 200 volts for 80 minutes. The remaining 20 μL of sample wasfrozen and stored at −70° C.

(k) The acrylamide-gel treated samples from step (i) were subjected toWestern blot analysis onto polyvinylidene fluoride (PVDF) membrane(Imobilon-P) for either 90 minutes at 40 volts or at 15 volts overnight(greater than 12 hours).

(l) The membrane was blocked in 5% milk protein and PBST for 45-60minutes.

(m) The treated samples were incubated with primary antibody for 1 hour,ICSM35B at 0.2 μg/mL in PBST. 25 mL volume was used for a singlemembrane.

(n) Blots were then developed according to the Storm® system for gel andblot analysis and blot imaging system for quantification.

Alternatively, a 5× PERASAFE™ Sterilant stock solution was prepared byadding 81 mg PERASAFE™ Sterilant powder to 1 mL of dd H₂O at 37° C. withvortex mixing to dissolve.

FIGS. 1 a and 1 b show the results of Western blot analyses fromProtocols B and C, respectively. Oxidation treatment alone (PERASAFE™Sterilant only—Protocol B) does not show prion destruction, butoxidation followed by protease treatment (Protocol C) is highlyeffective, the prion material disappearing by the ‘five minutes’ timepoint.

The ‘0 minutes’ treatment in FIG. 1 b shows less prion material thanmight be expected—this may be due to the procedures involved. Thistreatment is actually about 4 minutes, and each treatment is probablymore accurately regarded as being extended by 4 minutes so ‘5 minutes’is approx. 9 minutes, ‘10 minutes’ is approx. 14 minutes, and so on.Thus, 9 minutes of treatment appears to give destruction to undetectablelevels, without high temperatures and without autoclaving or use ofcaustic or corrosive chemical treatments.

Example 3 Decontamination of Surgical Surfaces

The prion-degrading materials (oxidizing agent and proteases) producedby Protocol B of Example 2 were carried out on infected homogenatedipped steel wire segments.

Steel wires (5 mm×0.15 mm) were incubated for 30 minutes with a 20%homogenate prepared from the brain of a CD1 mouse terminally sick withRocky Mountain Laboratories (RML) scrapie.

The wires were then treated according to the present invention andsignificant decontamination was observed for treatment under Protocol B.

A comparison was performed using Protocol B—use of oxidizing agent alone(PERASAFE™ Sterilant) was not effective.

Example 4 Prion-Decontamination Using an Oxidizing Agent (PERASAFE™Sterilant) in Combination with Prion-Degrading Proteases at 40° C.

The purpose of this experiment was to determine the efficacy ofdecontamination using combination of proteases with PERASAFE™ Sterilantat approximately 40° C.

Protocol A of Example 1 was followed unless otherwise noted. A singlereaction mixture containing 120 μL of 10% w/v brain homogenate in atotal volume of 200 μL was produced. Aliquots of 20 μL were removed atvarious time points, quenched and subjected to Western Blot analysis intoto. Thus, 12 μL equivalents of 10% w/v brain homogenate were analyzedat each time point. A high sensitivity protocol for the Western Blotdetection of PrP, with a known detection limit of 10 nL of a 10% w/vbrain homogenate was used. Thus complete destruction of PrP as evidencedby a total lack of immunoreactivity is equivalent to at least a 1,200fold reduction of infectivity.

The reaction was carried out in the presence of 1× PERASAFE™ Sterilantand 1% w/v SDS. Solid formulations of ALCALASE® and NEUTRASE® wereprepared as 100 mg/mL stock solutions in water and used at finalconcentrations of 3.16 mg/mL and 12.63 mg/mL, respectively.

The reaction mixture was incubated at 40° C. with 20 μL samples removedfor analysis at times 2, 10, 20, 30, 40 and 60 minutes. ResidualPrP^(Sc) was visualized by Western Blot detection using biotinylatedanti-PrP monoclonal antibody ICSM35 as the primary antibody as shown inFIG. 2. Column headings indicate M for Marker lane, C for control laneand the incubation times as the remaining lanes. Inspection of the blotin FIG. 2, indicates that complete destruction (>1,200 fold) wasachieved after 10 minutes at 40° C.

Example 5 Efficacy of Decontamination Using a Cell-Culture Assay ofPrion Infectivity

The efficacy of decontamination was assayed in a cell-culture assay (aScrapie Cell Assay; SCA). A prion-decontaminating composition containing1× PERASAFE™ Sterilant and 1% w/v SDS and a mixture of ALCALASE® andNEUTRASE® at final concentrations of 3.16 mg/mL and 12.36 mg/mL wasused. The composition was used at both 40° C. and 50° C. with a contacttime of 10 minutes.

In this assay stainless steel wires were coated with various dilutionsof brain homogenate from animals infected with the RML (Rocky MountainLaboratories) strain of prions (see, Example 3). The wires were theneither treated with the decontamination reagent or used untreated. Theywere incubated with a highly susceptible cell line (N2a-PK1; a murineneuroblastoma cell line) cloned to be highly susceptible to RML prions(see Klöhn et al., PNAS, 100: 11666-11671 (2003)). Following severalrounds of cell-growth and dilution the cells were filtered and subjectedto the MRC Prion Unit standard Scrapie Cell Assay (SCA; Klöhn et al.,supra) to quantify the number of tissue culture infectious unitspresent.

The dilution of RML proteins applied to the wires was plotted withrespect to the number of tissue culture infectious units (TC IU). Theresults of the assay are shown in FIG. 3. As can be seen from FIG. 3,complete destruction of contaminants (>109 fold) was achieved after 10minutes at 40° C. and 50° C. (decon at 40° C. and decon at 50° C.,respectively).

Example 6 Optimization of Sodium Dodecyl Sulfate Concentration withReagents Tested in Examples 4 and 5

The purpose of this example is to repeat the experiment as described inExample 4 with varying concentrations of surfactant (SDS) in the absenceof the oxidizing agent.

A basic protocol used raw 10% w/v brain homogenate. Individual reactionsmixtures containing 12 μL of 10% w/v brain homogenate in the presence ofALCALASE®/NEUTRASE® and varying concentrations (0, 0.5, 1.0, 1.5, and2.0% w/v) of SDS in a total volume of 20 μL were produced. Aliquots weresubjected to Western Blot analysis in toto. Thus, 12 μL equivalents of10% w/v brain homogenate were analyzed at each time point.

The reactions were carried out in the presence of 3.16 mg/mL ALCALASE®and 12.63 mg/mL NEUTRASE®. The reaction mixture was incubated at 50° C.for 10 minutes. Residual PrP^(Sc) was visualized by western blotdetection using biotinylated ICSM35 as the primary antibody. The highsensitivity protocol for the Western blot detection of PrP was used(with a known detection limit of 10 nL of a 10% w/v brain homogenate).Complete destruction of PrP as evidenced by a total lack ofimmunoreactivity is equivalent to at least a >1,200 fold reduction ofinfectivity, which is shown in FIG. 4. As show in FIG. 4, in the absenceof PERASAFE™ Sterilant, no condition gave complete destruction. As such,an SDS concentration of 1% w/v appears optimal.

Comparative Example A Optimization of ALCALASE® Concentration in theAbsence of PERASAFE™ Sterilant

A basic protocol used raw 10% w/v brain homogenate. Each reactionmixture was prepared to provide a total volume of 20 μL, containing 17μL of 10% w/v brain homogenate. The reaction mixtures contained 1% w/vSDS, 5 mg/mL NEUTRASE® and a range of ALCALASE® concentrations from 1.5mg/mL to 250 μg/mL. The reaction mixtures were incubated at 50° C. for10 minutes. A high sensitivity protocol for the Western Blot detectionof PrP, with a known detection limit of 10 nL of a 10% w/v brainhomogenate was used. Thus, complete destruction of PrP was evidenced bya total lack of immunoreactivity, which is equivalent to at least a1,700 fold reduction of infectivity.

Residual PrP^(Sc) was visualized by Western Blot detection usingbiotinylated ICSM35 as the primary antibody, which is shown in FIG. 5.The lanes are identified as M for marker lane, C for control and 1.5,1.25, 1.0, 0.75, 0.50, 0.25 and 0, based on the concentrations ofALCALASE® used, concentrations in mg/mL. As shown in FIG. 5, in theabsence of an oxidizing agent (i.e., PERASAFE™ Sterilant), the testedreagents showed low efficacy at 50° C. over 10 minutes.

Comparative Example B Optimization of NEUTRASE® Concentration in theAbsence of PERASAFE™ Sterilant

A basic protocol used raw 10% w/v brain homogenate. Each reactionmixture was prepared to provide a total volume of 20 μL containing 17 μLof 10% w/v brain homogenate. The reaction mixtures contained 1% w/v SDS,1.5 mg/mL ALCALASE® and a range of NEUTRASE® concentrations from 1.5g/mL to 250 μg/mL. The reaction mixtures were incubated at 50° C. for 10minutes. A high sensitivity protocol for the Western Blot detection ofPrP, with a known detection limit of 10 nL of a 10% w/v brain homogenatewas used. Thus, complete destruction of PrP was evidenced by a totallack of immunoreactivity, which is equivalent to at least a 1,700 foldreduction of infectivity.

Residual PrP^(Sc) was visualised by Western Blot detection usingbiotinylated ICSM35 as the primary antibody, which is shown in FIG. 6.The lanes are identified as M for marker lane, C for control and 1.5,1.25, 1.0, 0.75, 0.50, 0.25 and 0, based on the concentrations ofNEUTRASE® used, concentrations in mg/mL. As shown in FIG. 6, in theabsence of oxidizing agent (e.g., PERASAFE™ Sterilant), the testedreagents showed low efficacy at 50° C. over 10 minutes.

Example 7 Optimization of NEUTRASE®/ALCALASE® Concentrations in thePresence of PERASAFE™ Sterilan

The purpose of this experiment was to show that the rate of enzymeinclusion could be substantially reduced when used in combination withPERASAFE™ Sterilant.

A basic protocol used raw 10% w/v brain homogenate. Each reactionmixture was prepared to provide a total volume of 100 μL containing 60μL of 10% w/v brain homogenate. The reaction mixtures contained 1×PERASAFE™ Sterilant, 1% w/v SDS and range of ALCALASE® and NEUTRASE®concentrations (as measured by relative dilution rates; 2×, 4×, 8×, 16×,32×, and 64× dilutions). The reaction mixtures were incubated at 50° C.for 10 minutes and 40 μL of each reaction mixture was analyzed. A highsensitivity protocol for the Western Blot detection of PrP, with a knowndetection limit of 10 nL of a 10% w/v brain homogenate was used. Thus,complete destruction of PrP was evidenced by a total lack ofimmunoreactivity, which is equivalent to at least a 1,200 fold reductionof infectivity.

Residual PrP^(Sc) was visualized by Western Blot detection usingbiotinylated ICSM35 as the primary antibody, which is shown in FIG. 7.The lanes are identified as M for marker lane, C for control and 64, 32,16, 8, 4, 2 and 0, based on the dilution factors of proteaseconcentrations used. As shown in FIG. 7, the greatest destructionoccurred in the lane marked 2. This is a 2-fold dilution of the standardALCALASE®/NEUTRASE® concentrations (i.e., 1.58 mg/mL ALCALASE® and 6.32mg/mL NEUTRASE®). As a result, high levels of destruction can beachieved at enzyme concentrations 8-fold lower than previously tested incell-culture assays. The level of destruction is at least 1000-fold.Effective decontamination at 50° C. for 10 minutes in the presence ofPERASAFE™ Sterilant with 1% w/v SDS and enzyme concentrations of 400μg/mL and 1.6 mg/mL ALCALASE® and NEUTRASE®, respectively.

Example 8 Further Optimization of ALCALASE®/NEUTRASE® Concentrations inthe Presence of PERASAFE™ Sterilant

A basic protocol used raw 10% w/v brain homogenate. Each reactionmixture was prepared to provide a total volume of 100 μL containing 60μL of 10% w/v brain homogenate. The reaction mixtures contained 1×PERASAFE™ Sterilant, 1% w/v SDS and range of ALCALASE® and NEUTRASE®concentrations at various dilutions (4 [790 μg/mL ALCALASE®+3.16 mg/mLNEUTRASE®], 6.7 [474 μg/mL ALCALASE®+1.89 mg/mL NEUTRASE®], 8.3 [379μg/mL ALCALASE®+1.52 mg/mL NEUTRASE®], 10 [316 μg/mL ALCALASE®+1.26mg/mL NEUTRASE®], and 12.5 [253 μg/mL ALCALASE®+1.01 mg/mL NEUTRASE®].The reaction mixtures were incubated at 50° C. for 10 minutes and 40 μLof each reaction was analyzed. A high sensitivity protocol for theWestern Blot detection of PrP, with a known detection limit of 10 nL ofa 10% w/v brain homogenate was used. Thus complete destruction of PrPwas evidenced by a total lack of immunoreactivity, which is equivalentto at least a 1,200 fold reduction of infectivity.

Residual PrP^(Sc) was visualised by Western Blot detection usingbiotinylated ICSM35 as the primary antibody, which is shown in FIG. 8.The lanes are identified as M for marker lane, C for control and 4, 6.7,8.3, 10, and 12.5, based on the dilution factors of proteaseconcentrations used. As shown in FIG. 8, high levels of destruction canbe achieved at ALCALASE® and NEUTRASE® concentrations 10-fold lower thanpreviously tested in cell-culture.

Comparative Example C Testing of Individual Components that ComprisePERASAFE™ Sterilant with SDS and Enzymes

A basic protocol used raw 10% w/v brain homogenate. Each reactionmixture was prepared to provide a total volume of 20 μL containing 16 μLof 10% w/v brain homogenate. The reaction mixtures contained 1% w/v SDS,750 g/mL ALCALASE®, 3 mg/mL NEUTRASE® plus various components ofPERASAFE™ Sterilant.

Lane Component (a) Stabilizers/Corrosion Inhibitors (191 μg/mL) (b)Surfactant (165 μg/mL) (c) Organic Acid (3.36 mg/mL) (d) Activator (4.44mg/mL) (e) Oxidizing Agent (8.03 mg/mL) (f) Water (control)

The reactions were incubated at 50° C. for 10 minutes and 40 μL of eachreaction was analyzed. A high sensitivity protocol for the Western Blotdetection of PrP, with a known detection limit of 10 nL of a 10% w/vbrain homogenate was used. Thus, complete destruction of PrP wasevidenced by a total lack of immunoreactivity, which is equivalent to atleast a 1,600 fold reduction of infectivity.

Residual PrP^(Sc) was visualised by Western Blot detection usingbiotinylated ICSM35 as the primary antibody, as shown in FIG. 9. Thelanes are identified as M for marker lane, C for control and a-f, basedon the component identified above. FIG. 9 shows no single componentproduced levels of destruction comparable to those observed with activePERASAFE™ Sterilant (e). It appears that efficacy is result of theoxidative activity provided by the peracetic acid generated fromTAED/sodium perborate. However, when combined with proteases in thecompositions of this invention, significantly improved prion destructionis achieved.

Example 9 Repeat of the Optimization of ALCALASE®/NEUTRASE®Concentrations in the Presence of PERASAFE™ Sterilant

The experiment described in Example 8 was repeated to confirm a 10-foldreduction in enzyme levels in the formulation was effective when enzymeswere combined with oxidizing agent.

A basic protocol used raw 10% w/v brain homogenate. Each reactionmixture was prepared to provide a total volume of 100 μL containing 60μL of 10% w/v brain homogenate. The reaction mixtures contained 1×PERASAFE™ Sterilant, 1% w/v SDS and range of ALCALASE® and NEUTRASE®concentrations (4 [790 μg/mL ALCALASE®+3.16 mg/mL NEUTRASE®], 6.7 [474μg/mL ALCALASE®+1.89 mg/mL NEUTRASE®], 8.3 [379 μg/mL ALCALASE®+1.52mg/mL NEUTRASE®], 10 [316 μg/mL ALCALASE®+1.26 mg/mL NEUTRASE®], and12.5 [253 μg/mL ALCALASE®+1.01 mg/mL NEUTRASE®]. The reaction mixtureswere incubated at 50° C. for 10 minutes and 40 μL of each reaction wasanalyzed. A high sensitivity protocol for the Western Blot detection ofPrP, with a known detection limit of 10 nL of a 10% w/v brain homogenatewas used. Thus, complete destruction of PrP was evidenced by a totallack of immunoreactivity, which is equivalent to at least a 1,200 foldreduction of infectivity.

Residual PrP^(Sc) was visualised by Western Blot detection usingbiotinylated ICSM35 as the primary antibody, as shown in FIG. 10. Thelanes are identified as M for marker lane, C for control and 4, 6.7,8.3, 10, and 12.5, based on the dilution factors of proteaseconcentrations used. FIG. 10 shows high levels of destruction can beachieved at ALCALASE® and NEUTRASE® concentrations at least 10-foldlower than previously tested in cell culture. FIG. 10 confirms theprevious findings of Example 8.

Example 10 Further Testing of Individual Components that ComprisePERASAFE™ Sterilant with SDS and Enzymes+Redox Active Combinations

A basic protocol used raw 10% w/v brain homogenate. Each reactionmixture was prepared to provide a total volume of 20 μL containing 13 μLof 10% w/v brain homogenate. The reaction mixtures contained 1% w/v SDS,300 μg/mL ALCALASE®, 1.2 mg/mL NEUTRASE® plus various components orconcentrations of PERASAFE™ Sterilant.

Lane Component (a) PERASAFE ™ Sterilant (16.2 g/L) (1X) (b) PERASAFE ™Sterilant (8.1 g/L) (0.5X) (c) PERASAFE ™ Sterilant (3.24 g/L) (0.2X)(d) Stabilizers/Corrosion Inhibitors (191 μg/mL) (e) Sodium Perborate +Activator (8.03 mg/mL)

The reactions were incubated at 50° C. for 10 minutes and 40 μL of eachreaction was analyzed. A high sensitivity protocol for the Western Blotdetection of PrP, with a known detection limit of 10 nL of a 10% w/vbrain homogenate was used. Thus, complete destruction of PrP wasevidenced by a total lack of immunoreactivity, which is equivalent to atleast a 1,300 fold reduction of infectivity.

Residual PrP^(Sc) was visualised by Western Blot detection usingbiotinylated ICSM35 as the primary antibody, as shown in FIG. 11. Thelanes are identified as M for marker lane, C for control and a-e, basedon the component identified above. FIG. 11 shows high levels ofdestruction were confirmed with 1× PERASAFE™ Sterilant in the presenceof 1% w/v SDS, 300 μg/mL ALCALASE® and 1.2 mg/mL NEUTRASE®. It appearsthat a reduced inclusion rate of 0.5× PERASAFE™ Sterilant may providesufficient levels of decontamination.

Example 11 Comparing the Efficacy of Decontamination by the 1× PERASAFE™Sterilant based Decontamination Composition at 40° C. and 50° C.

The purpose of the following experiment was to compare the efficacy ofdecontamination by the decontamination composition (1× PERASAFE™Sterilant+1% w/v SDS+300 μg/mL ALCALASE®+1.2 mg/mL NEUTRASE®) at 40° C.and 50° C.

A basic protocol used raw 10% w/v vCJD brain homogenate. A reactionmixture containing 60 μl of 10% w/v brain homogenate in a total volumeof 100 μl was used for each temperature. The reaction mixtures containedan equivalent formulation of the decontamination mixture [1× PERASAFE™Sterilant and 1% w/v SDS+300 μg/mL ALCALASE®+1.2 mg/mL NEUTRASE®].Aliquots of 20 μL were removed at various time points, quenched byadding to 2×SDS loading buffer and freezing in liquid nitrogen. Allreaction mixtures were thawed and boiled before subjecting to WesternBlot detection for residual PrP^(Sc) using biotinylated ICSM35 as theprimary antibody.

The reaction mixtures (40° C. vs. 50° C.) were sampled at time 0, 2, 5,10 and 20 minutes and the results are shown in FIG. 12, with the sampletimes as the lane labels. C is for control. The levels ofimmunoreactivity in FIG. 12 were quantified by densitometry and plottedas function of time in FIG. 13. The kinetic curves describing the lossof PrP, % Immunoreactivity Remaining, with respect to time in minutes,were fitted to double exponential decay functions and the resultssuperimposed as lines through the data in FIG. 13. A slight reduction inrate of destruction is observed at 40° C. than 50° C. However, very highlevels of destruction are achieved after 10 min incubation time at bothtemperatures.

Example 12 Comparison of Oxidizing Agents with PERASAFE™ Sterilant asAdditives to Composition to Decontaminate Infected Entities

The purpose of the following examples was to compare chemical oxidizingagents for use in a decontamination composition comprising SDS (1% w/v)and two prion-degrading proteases (300 μg/mL ALCALASE® and 1.2 mg/mLNEUTRASE®). The oxidizing agents included NaDCC (sodiumdichloroisocyanurate), PROXITANE® (a peracetic acid-based disinfectantavailable from Solvay Chemicals, Brussels, Belgium), VIRKON® SDisinfectant (a disinfectant comprising potassium peroxymonosulfate andNaCl, sodium dodecylbenzene sulfonate, and sulfamic acid), hypochloritesolution (i.e., bleach), and PERASAFE™ Sterilant.

A basic protocol used raw 10% w/v vCJD brain homogenate. Each reactionmixture was prepared using 60 μL of 10% w/v brain homogenate in a totalvolume of 100 μL. Constant concentrations of SDS and enzymes [1% w/v SDSand 300 μg/mL ALCALASE®+1.2 mg/mL NEUTRASE®] were used in all of thereaction mixtures and all reaction mixtures were incubated for 10minutes at 50° C.

Reaction A contained 16.2 mg/mL standard PERASAFE™ Sterilant.

Reaction B contained 13.0 mg/mL standard PERASAFE™ Sterilant.

Reaction C contained 13.0 mg/mL standard PERASAFE™ Sterilant.

Reaction H contained a 1:70 dilution of hypochlorite solution (i.e.,bleach), (pH 8.0).

Reaction N contained 3.34 mg/mL NaDCC.

Reaction P contained 30 mg/mL PROXITANE® (pH 6.5).

Reaction V contained 20 mg/mL VIRKON® S (pH 6.0).

Aliquots of 20 μL were removed after 10 minutes incubation and quenchedby adding to 2×SDS loading buffer and freezing in liquid nitrogen. Allreaction mixtures were thawed and boiled before subjecting to WesternBlot detection for residual PrP^(Sc) using biotinylated ICSM35 as theprimary antibody. The results are shown in FIG. 14. The lanes on the gelcorrespond to the reactions listed above.

As shown in FIG. 14, other oxidizing agents are effective when used asthe oxidizing agent in a decontamination composition comprisingsurfactant and the two different prion-degrading proteases. The levelsof immunoreactivity in FIG. 14 were quantified by densitometry and aredisplayed as a percentage of the control value, % Immunoreactivity, inFIG. 15.

1. An aqueous prion-degrading composition comprises an oxidizing agent,at least one protease, and a surfactant that can be used for effectiveprion degradation.
 2. The composition of claim 1 comprising a firstprotease and a second protease.
 3. The composition of claim 1 whereinthe oxidizing agent is selected from the group consisting of peroxide,persalt, peracids, persulfates, peroxyphthalates, organic chlorines,chlorine dioxide, and stable mixtures thereof.
 4. The composition ofclaim 3 wherein the oxidizing agent comprises peroxide, a modifiedperoxide, a peracid, such as peracetic acid, or mixtures thereof.
 5. Thecomposition of claim 4wherein the oxidizing agent is a peroxygen system,which generates peracetic acid and peracetic anions in situ upondissolution.
 6. The composition of claim 1 wherein the protease isselected from the group consisting of NEUTRASE®, ALCALASE®, PRONASE®,Proteinase K, and combinations thereof.
 7. The composition of claim 2wherein the first and second proteases are NEUTRASE® and ALCALASE®. 8.The composition of claim 1 wherein the surfactant is an ionic surfactantselected from the group consisting of sodium dodecylbenzene sulfonate,lauryl ether sulfate, ethylene oxide/propylene oxide alkyl phenolcondensate, polyglycol ether of fatty alcohols, fatty acid ethyleneoxide condensate, polyglycol ether of alkyl phenols, fatty alcoholethoxylate, sodium lauryl ether sulfate, sodium dodecyl sulfate, andcombinations of two or more thereof.
 9. The composition of claim 8wherein the surfactant is sodium dodecyl sulfate (SDS), sodiumtaurodeoxycholate hydrate, sodium 1-octanesulfonate monohydrate, lithiumdodecyl sulphate, N-lauroylsarcosine sodium salt, or a combination oftwo or more thereof.
 10. The composition of claim 9 wherein thesurfactant is sodium dodecyl sulfate (SDS).
 11. A method for priondecontamination or disinfection is provided which comprises insequential order or simultaneously: (a) contacting a prion-contaminatedentity with at least one surfactant; (b) contacting the entity with anoxidizing agent, and (c) contacting the entity with at least oneprotease.
 12. The method of claim 11 further comprising (d) contactingthe entity with a first protease and a second protease.
 13. The methodof claim 12 steps (a), (b), (c) and (d) are performed sequentially. 14.The method of claim 12 wherein steps (a), (b), (c) and (d) are performedsimultaneously.
 15. The method of claim 12 wherein the first and secondproteases are selected from the group consisting of NEUTRASE®,ALCALASE®, PRONASE®, Proteinase K, and combinations thereof.
 16. Themethod of claim 12 wherein the first and second proteases are NEUTRASE®,and ALCALASE®.
 17. The method of claim 12 wherein the prion-contaminatedentity has a solid surface or is a biological fluid.
 18. The method ofclaim 12 wherein the prion-contaminated entity is selected from thegroup consisting of a biological waste, equipment used in foodprocessing equipment, an enclosure used to house animals, a medicalinstrument, a dental instrument, and countertops.
 19. The method ofclaim 12 wherein the prion-contaminated entity is a prion-contaminatedmedical instrument.
 20. The method of claim 12 further comprisingautoclaving after the contacting steps.
 21. A kit which comprises: (a) afirst reagent comprising an oxidizing agent; (b) a second reagentcomprising at least one protease; and (c) a third reagent comprising asurfactant.
 22. The kit of claim 21 wherein the second reagent in thekit comprises at least two different proteases.
 23. The kit of claim 22wherein the at least two proteases are selected from the groupconsisting of NEUTRASE®, ALCALASE®, PRONASE®, and Proteinase K.
 24. Thekit of claim 22 wherein the first, second, and third reagents areprovided individually as solids.